Human BCDF, which is a factor that differentiates human B cells into antibody-producing cells, had its cDNA cloned in 1986 (Nature, 324, 73 (1986)). Later, this substance has come to be called B-cell stimulatory factor-2 (BSF-2) or interleukin 6 (IL-6), and its properties have been clarified on the whole (molecular weight: about 21,000; isoelectric point: about 6.2). It has been also reported that human BCDF showed various bioactivities. Among those activities, there are included medicinal activities such as stem blood cell proliferating action, the action to differentiate their precursor cells into platelets, and the action to differentiate B lymphocytes into antibody-producing cells. Thus, utilization of human BCDF is expected in the field of medicine, as, e.g., a medicinal preparation for making up for the decrease of hemocytes resulting from the use of a carcinostatic agent or bone marrow transplantation, or a medicinal preparation for reinforcing the vaccine action.
The present inventors have already disclosed some methods of simply and effectively separating pure human BCDF from human BCDF cultured broths obtained by using, as production host, an Escherichia coli having cDNA for human BCDF integrated therein (see Japanese Patent Applications Kokai Nos. Hei 1-83094, Hei 1-300898 and Hei 2-186996). These methods, however, are not always satisfactory for removing impurities such as analogs of human BCDF, said impurities having been recognized in the course of study of the techniques for separating pure human BCDF from human BCDF cultured broths. Also, the degree of purification achievable by these methods, although satisfactory for laboratory research, is still unacceptable for the production of human BCDF to be used for medicine. For human BCDF to be used as medicine, it is necessary to establish a practical purification process on the premise that a medicinal base free of any substance harmful to the human body is industrially produced at a small cost and with a high purity sufficient for safe administration to man.
It is generally known that when a physiologically active protein is produced by using a recombinant DNA in E. coli as host, there may occur contamination with an analog protein partially differing in primary structure from the natural type or an analog protein differing in disulfide linkage from the natural type. Also, as is known in connection with interleukin 2 and interferon, there may be cases where a desired protein is contaminated with a high-molecular weight substance formed as a result of inter molecular association by non-covalent bond, said desired protein being normally supposed to exist as a monomer. Further, there are cases where an analog is produced as a result of cleavage or partial modification of a desired protein or variation of a desired protein in primary structure or stereostructure in the process of purification. These analogs involve the danger of causing a harmful immunoreaction by producing an unnecessary antibody when they are administered repeatedly to the human body. The existence of inter molecular aggregates of human BCDF was discovered for the first time by the present inventors, as described below, and therefore its separation method has not been known.
Further, there has not been disclosed a combination of the purification techniques according to which
(1) formation of denatured proteins from human BCDF may be minimized, which denatured proteins may cause lowering of the physiological activities of human BCDF, and
(2) proteins derived from the microorganism which may be harmful to the human body may be removed,
(3) whereby a highly pure human BCDF cleared of exothermic substances (such as endotoxin) derived from the microorganism or included in the course of the purification process, along with the above-mentioned techniques for removing the analogs.
Lymphokines such as human BCDF normally works in vivo at a concentration of as little as about several pg/ml, but for the purpose of treating a lymphokine as a medicinal base, it is desirable to treat it at a high concentration of as much as about several mg/ml. The reasons are that 1) since high dosage toxicity tests can be conducted, safety evaluation of the lymphokine can be made with higher accuracy, 2) when a medicinal preparation is made from a base, it can be easily adjusted to an appropriate concentration for administration even if various adjuvants are added, which, in turn, means that it is easy to make a medicinal preparation, and 3) since the purification process can be carried out on a small scale, it becomes possible to simplify the operation and apparatus concerned. Lymphokines are, however, generally highly hydrophobic, and the risk of inter molecular association, precipitation or denaturation increases as the concentration increases, so that in the purification of human BCDF according to the present invention, it was necessary to elaborate the purification conditions, different from the purification under the low concentration conditions for cultures of cells, etc.